Design and Characterization of a 52K SNP Chip for Goats

The success of Genome Wide Association Studies in the discovery of sequence variation linked to complex traits in humans has increased interest in high throughput SNP genotyping assays in livestock species. Primary goals are QTL detection and genomic selection. The purpose here was design of a 50–60,000 SNP chip for goats. The success of a moderate density SNP assay depends on reliable bioinformatic SNP detection procedures, the technological success rate of the SNP design, even spacing of SNPs on the genome and selection of Minor Allele Frequencies (MAF) suitable to use in diverse breeds. Through the federation of three SNP discovery projects consolidated as the International Goat Genome Consortium, we have identified approximately twelve million high quality SNP variants in the goat genome stored in a database together with their biological and technical characteristics. These SNPs were identified within and between six breeds (meat, milk and mixed): Alpine, Boer, Creole, Katjang, Saanen and Savanna, comprising a total of 97 animals. Whole genome and Reduced Representation Library sequences were aligned on >10 kb scaffolds of the de novo goat genome assembly. The 60,000 selected SNPs, evenly spaced on the goat genome, were submitted for oligo manufacturing (Illumina, Inc) and published in dbSNP along with flanking sequences and map position on goat assemblies (i.e. scaffolds and pseudo-chromosomes), sheep genome V2 and cattle UMD3.1 assembly. Ten breeds were then used to validate the SNP content and 52,295 loci could be successfully genotyped and used to generate a final cluster file. The combined strategy of using mainly whole genome Next Generation Sequencing and mapping on a contig genome assembly, complemented with Illumina design tools proved to be efficient in producing this GoatSNP50 chip. Advances in use of molecular markers are expected to accelerate goat genomic studies in coming years.     

Analysis of the Complete Nucleotide Sequence of the Tetracycline-Resistance Transposon Tn10

An analysis of the complete nucleotide sequence of the composite tetracycline-resistance transposon Tn10 (9147 bp) from the Salmonella typhi conjugative plasmid R27 is presented. A comparison of the protein sequences from IS10-right and IS10-left transposases has identified four amino acid differences. These residues appear to play an important role in normal transposase function and may account for the differences in exhibited transposition activities. The tetracycline determinants encoded by this version of Tn10 share >99% identity with those of Tn10^R100, demonstrating the conservation that exists between these transposons. A previously uncharacterized 3000-bp region of Tn10 contains four putative open reading frames. One of these open reading frames shares 55% identity with the glutamate permease protein sequence from Haemophilus influenzae although it was unable to complement an Escherichia coli glutamate permease mutant, with which it shares 51% identity. The three remaining putative open reading frames are arranged as a discrete genetic unit adjacent to the glutamate permease homolog and are transcribed in the opposite direction. Two of these open reading frames are homologous with Bacillus subtilis proteins of unknown functions while the other has no homologs in the database. The presence of an aminoacyl-tRNA synthetase class II motif in one of these open reading frames in combination with the glutamate permease homolog allows us to postulate that this region of Tn10 could once have played a role in amino acid metabolism.     

Tissue distribution and mode of DNA methylation in mice by methyl methanesulphonate and N-methyl-N' -nitro-N-nitrosoguanidine: lack of thymic lymphoma induction and low extent of methylation of target tissue DNA at 0-6 of guanine.

The methylating agents methyl methanesulphonate (MMS) and N-methyl N'-nitro-N-nitrosoguanidine (MNNG), administered by single i.p. injection in mice failed to yield thymic lymphoma at doses around 60% of the LD50 values, in contrast to MNUA which gives a high yield of tumours by this route. Comparison of the tissue distribution and mode of DNA methylation by these agents showed a positive correlation with ability to methylate the 0-6 atom of guanine in DNA of the target tissues thymus and bone marrow and tumorigeneis. MMS gave a low yield of this product due to its relatively low Sn1 reactivity but was able to methylate DNA extensively at other sites in the target tissues and other organs examined. MNNG despite its ability to methylate 0-6 of guanine in DNA in vitro to the same relative extent as the potent carcinogen MNUA, methylated DNA of thymus and bone marrow to a very small extent in vivo but was able to methylate DNA in certain other tissues nearer the site of i.p. injection. These findings contrast with the general relatively extensive methylation of 0-6 of guanine in DNA of the target tissues and other organs by N-methyl-N-nitrosourea (MNUA).     

Human C5a modulates monocyte Fc and C3 receptor expression

FcIgG and C3 (CR1 and CR3) receptors are responsible for binding opsonized particles, phagocytosis, and immune adherence reactions by circulating and tissue-fixed mononuclear phagocytes. Alterations in the expression of these receptors may thus significantly influence the function of these cells. Because chemoattractants have been shown to both recruit and modulate the function of monocytes, this study specifically examines the effects of human C5a and N-formyl-methionyl-leucyl-phenyl-alanine (FMLP) on human peripheral blood monocyte FcIgG and C3 receptor expression in vitro. Adherent, elutriator-purified monocytes were incubated with C5a (10(-7) to 10(-10) M) or FMLP (10(-5) to 10(-10) M) for 30 min at 37 degrees C, and FcIgG receptor expression was assessed by rosetting with sheep erythrocytes sensitized with limiting dilutions of IgG. Human C5a caused dose-related increases in Fc rosettes of 28% at 10(-9) M, 63% at 10(-8) M, and 167% at 10(-7) M (p less than 0.01). In contrast, no significant increases in monocyte Fc receptor expression were induced by FMLP. Similar rosetting experiments were performed with sheep erythrocytes opsonized with limiting amounts of human C3b to assess C3b receptor expression on adherent human monocytes stimulated with C5a (10(-7) to 10(-10) M) or FMLP (10(-6) to 10(-9) M) for 30 min at 37 degrees C. Again, human C5a caused dose-related increases in monocyte C3b rosette formation; at 10(-8) M and 10(-7) M concentrations of C5a, these increases equaled 119% and 196%, respectively (p less than 0.05). In these experiments, 10(-6) M FMLP also caused a significant increase of 110% in monocyte C3b rosette formation (p less than 0.05). Modulation of monocyte cell surface receptors by human C5a or FMLP was also examined by measuring cell fluorescence and side scatter by dual channel flow cytometry after staining normal leukocytes in citrated venous blood with receptor-specific monoclonal antibodies. These flow cytometric studies demonstrated that both C5a and FMLP induce dose-related increases in CR1 (C3b receptor) and CR3 (iC3b receptor) expression in both monocytes and neutrophils.(ABSTRACT TRUNCATED AT 400 WORDS)     

Human dermal microvascular endothelial but not human umbilical vein endothelial cells express CD36 in vivo and in vitro.

CD36 is an 88-kDa glycoprotein that has been identified on platelets, monocytes, and some endothelial cells. Experimental evidence suggests that CD36 mediates the binding of Plasmodium falciparum-infected RBC to a variety of cells, and therefore may play a role in the vascular complications associated with malaria. Additionally, CD36 may also bind the extracellular matrix proteins thrombospondin and collagen. Human umbilical vein endothelial cells have been used in in vitro models examining the binding of P. falciparum RBC to endothelial cells, but they do not consistently express cell surface CD36. Inasmuch as human dermal microvascular endothelial cells (HDMEC) differ in a variety of ways from large vessel endothelial cells, we have examined HDMEC for cell surface expression of CD36 in vivo and in vitro. Direct immunofluorescence of skin showed bright staining of HDMEC with antibody recognizing CD36 and flow cytometric analysis of cultured HDMEC revealed cell surface expression. In contrast, large vessel endothelial cells were not stained with antibody recognizing CD36 in vivo and cultured cells derived from umbilical vein failed to express cell surface CD36 in vitro. Western immunoblots of lysates of HDMEC but not human umbilical vein endothelial cells demonstrated an 88-kDa protein that comigrated with CD36 from platelets. Functional studies demonstrated that adherence of PRBC to HDMEC was inhibited up to 66% by mAb recognizing CD36. Furthermore, the expression of CD36 on HDMEC was increased in a dose- and time-dependent manner by IFN-gamma, and was decreased by protein kinase C agonists. These data demonstrate that HDMEC express functionally active CD36 and this expression can be positively and negatively regulated by soluble factors. This study demonstrates that HDMEC are useful in the study of CD36-mediated binding of PRBC to endothelial cells in vitro and provides further evidence of distinct phenotypic differences between HDMEC and large vessel endothelial cells.     

Design Guidelines for Deadlock-Handling Strategies in Flexible Manufacturing Systems

Deadlock-free operation of flexible manufacturing systems (FMSs) is an important goal of manufacturing systems control research. In this work, we develop the criteria that real-time FMS deadlock-handling strategies must satisfy. These criteria are based on a digraph representation of the FMS state space. Control policies for deadlock-free operation are characterized as partitioning cuts on this digraph. We call these structural control policies (SCPs) because, to avoid deadlock, they must guarantee certain structural properties of the subdigraph containing the empty state; namely, that it is strongly connected. A policy providing this guarantee is referred to as correct. Furthermore, an SCP must be configurable and scalable; that is, its correctness must not depend on configuration-specific system characteristics and it must remain computationally tractable as the FMS grows in size. Finally, an SCP must be efficient; that is, it must not overly constrain FMS operation. We formally develop and define these criteria, formulate guidelines for developing policies satisfying these criteria, and then provide an example SCP development using these guidelines. Finally, we present an SCP that guarantees deadlock-free buffer space allocation for FMSs with no route restrictions.     

Some chemical aspects of dose-response relationships in alkylation mutagenesis

Alkylation of DNA can lead to induction of potentially miscoding groups (promutagenic) or potentially template-inactivating groups (lethal). The proportions of these are found to vary with the chemical nature of the alkylating agent. Agents of low Swain and Scott s factor (or those tending to Ingold's S_Ni type) react relatively more extensively at O-atom sites in DNA, and yield relatively more of the miscoding O₆-alkylguanine residues. Phosphotriester formation is also relatively more extensive with S_Ni agents.Inactivation of DNA can result from depurinations, strand breakage, and cross-linkage.Both promutagenic and lethal lesions are subject to repair; 3 principal enzymatic systems appear to exist; one for excision and repair of cross-links or aralkyl groups resembles the uvr system; others for repair of single-strand breaks parallel repair of X-ray-induced breaks (exr, rec systems); another, less well defined at present, recognizes certain methylated bases, and depurinated sites (probably Goldthwait's endonuclease II).These factors can be shown to influence dose-response in alkylation mutagenesis. This, broadly, can be classified as linear with the promutagenic group-inducing or directly miscoding agents, and is independent of cytotoxicity; whereas with other agents non-linear response parallels the occurrence of “shouldered” survival curves, and reflects mutation induction by “repairs errors”.Additionally, alkylation of cellular constituents other than DNA, e.g. repair enzymes, may influence dose response, and will again depend on chemical reactivity of the agent.     

Increased urinary excretion of labelled deoxyribonucleosides in rats with stable pre-label led dna treated with methyl methanesulphonate or nitrogen mustard

After the DNA of newborn female rats had been labelled by repeated injections of [¹⁴C]orotate (totalling 36 μCi) during the first 3 weeks of life, approximately 1 000 000 dpm were found in the DNA of the liver, lungs, kidneys, gut, brain, heart and spleen of 8-week-old rats. Methyl methanesulphonate (MMS) (80 mg/kg) and di-(2-chloroethyl)methylamine (HN2) (5 mg/kg) injection increased the amount of ¹⁴C-labelled DNA pyrimidine nucleosides excreted in the urine to 5000 dpm from 350 dpm before injection. The effect on RNA products was much less marked.